This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Trypanosoma brucei is a unicellular parasitic protozoan that causes African sleeping sickness in humans and Ngana in cattle and other livestock. The parasite has a complex lifecycle with transitions in cell shape during both the human and the tse tse fly infection. Responsible for the maintenance and restructuring of these cell shapes is a complex microtubule cytoskeleton, consisting of a sub-pellicular microtubule array, the flagellar axoneme, basal bodies and the mitotic spindle. The sub-pellicular microtubule array is a complete corset of equally interspaced filaments, cross-linked to each other and to the plasma membrane. The microtubules within this array are hyper-stable and resist detergent extraction. We wonder how the stable sub-pellicular microtubule array allows for the constant rearrangement of cell shape that is caused by flagellar beating, cell growth and division. How is microtubule cross-bridging achieved and maintained? How is the new flagellar pocket inserted into the corset without causing gaps in between the filaments? Constant flagellar beating is essential for the viability of these cells, and the correct cell shape (i.e. life cycle stage) is important for transfer or the parasite between humans and the tse tse fly. Therefore, understanding the microtubule cytoskeleton and its associated proteins might reveal important novel drug targets. We will use electron tomography to yield maps of this cell's internal organization and its changes during the cell cycle, and analyze the detailed protein arrangement associated with the microtubule cytoskeleton using cryo-electron tomography.